In the purification of biological streams, the goal is to capture one or more desired proteins from a liquid stream that contains a variety of components such as other proteins, lipids, aggregated proteins from the cells, media proteins, carbohydrates and colloids from cell walls, DNA, DNA/protein complexes and the like. A number of purification steps may be used but general revolve around one or both of affinity chromatography and ultrafiltration (UF) systems.
The liquid stream is generally derived from a batch of lysed cells that have been engineered to produce the desired protein. Most of the cell components are removed from the liquid by filtration or centrifugation before chromatography and/or ultrafiltration. However a surprisingly large amount, especially of small or soluble constituents remain in the liquid as it is applied to the purification system. Many of these compete with the desired protein and bind to the affinity material in the column or clog the ultrafilter.
In affinity chromatography, some of the constituents bind preferentially so over that of the desired protein reducing yields. Others irreversibly bind to the affinity material reducing not only yields but overall capacity especially on subsequent uses of the media.
Most of these non-desired species are known as non-specific binding species and the problem has been called non-specific binding (NSB).
Various methods have been attempted to reduce NSB, both to increase yield initially and to maintain capacity over time for a column and to maintain a desired rate of flux and extend the useful life of a UF system.
Generally, a column is washed several times between the affinity capture stage and the elution stage with various buffers at different pHs and/or with different chemical washes in an attempt to remove the NSB species. Likewise the use of various cleaning regiments have been employed after use to try and remove as much of the NSB species from the media as possible to maintain yield and capacity values.
None of these have proven to greatly effective especially with the most commonly used affinity media in the biopharmaceutical industry. In part this is due to the affinity ligand itself Protein A. It being a protein is susceptible to denaturing under various conditions that other wise could be used to reduce NSB such as heat, high caustic concentrations and the like.
UF systems are cleaned after the system has become plugged enough to create too high a back pressure or to reduce the flux below a given level. Its filters are then cleaned with caustic solutions before reuse.
While the washing with caustic often results in a membrane free of NSB species, it does nothing to extending the life of a filter during filtration. The problem of fouling still exists.
What is needed is a means for reducing NSB to a lower level before the liquid ever reaches the purification step. The present invention provides such an invention.